EP4062976A1 - Acide carbalysophosphatidique - Google Patents

Acide carbalysophosphatidique Download PDF

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Publication number
EP4062976A1
EP4062976A1 EP20890320.3A EP20890320A EP4062976A1 EP 4062976 A1 EP4062976 A1 EP 4062976A1 EP 20890320 A EP20890320 A EP 20890320A EP 4062976 A1 EP4062976 A1 EP 4062976A1
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Prior art keywords
group
lpa
linear
carbon atoms
group containing
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EP20890320.3A
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German (de)
English (en)
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EP4062976A4 (fr
Inventor
Kimiko Murofushi
Mari GOTOH
Keiko Fukasawa
Junken Aoki
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MUROFUSHI, KIMIKO
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Ochanomizu University
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/66—Phosphorus compounds
    • A61K31/665—Phosphorus compounds having oxygen as a ring hetero atom, e.g. fosfomycin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic Table
    • C07F9/02—Phosphorus compounds
    • C07F9/28—Phosphorus compounds with one or more P—C bonds
    • C07F9/38—Phosphonic acids [RP(=O)(OH)2]; Thiophosphonic acids ; [RP(=X1)(X2H)2(X1, X2 are each independently O, S or Se)]
    • C07F9/3804—Phosphonic acids [RP(=O)(OH)2]; Thiophosphonic acids ; [RP(=X1)(X2H)2(X1, X2 are each independently O, S or Se)] not used, see subgroups
    • C07F9/3808—Acyclic saturated acids which can have further substituents on alkyl

Definitions

  • the present invention relates to carbalysophosphatidic acid having autotaxin inhibitory activity, LPA receptor-activating action, and ERK-phosphorylating action.
  • Non-Patent Document 1 This substance has been found to be a substance, in which hexadecanoic acid containing cyclopropane binds to position sn-1 of a glycerol backbone and phosphate group binds to positions 2 and 3 thereof via a cyclic ester bond.
  • PHYLPA Since this substance is an LPA-like substance derived from Physarum, it was named as PHYLPA.
  • PHYLPA has characteristic fatty acid at position sn-1. Thus, a derivative thereof was chemically synthesized by substituting the characteristic fatty acid with common fatty acid, and the activity thereof was then examined. As a result, it was demonstrated that PHYLPA suppresses cell proliferation, and it was revealed that the action of PHYLPA to suppress cell proliferation is caused by the cyclic phosphoric acid groups at position 2 and at position 3.
  • LPA analogs having such cyclic phosphoric acid groups are collectively referred to as "cyclic phosphatidic acid (cPA).
  • Patent Documents 1 and 2 there have been reports about neurotrophin action and application thereof to neurodegenerative disease (Patent Documents 1 and 2), suppression of proliferation of cancer cells and infiltration/metastasis thereof (Patent Document 3), analgesic action (Patent Document 4), application to atopic dermatitis (Patent Document 5), action to suppress demyelination of nerve axons (Patent Document 6), and the like.
  • Non-Patent Document 1 Murakami-Murofushi, K., et al., J. Biol. Chem. 267, 21512-21517 (1992 )
  • the present inventors have discovered and identified carbalysophosphatidic acid as a novel analog of carbacyclic phosphatidic acid.
  • the present inventors have further demonstrated that the above-described carbalysophosphatidic acid has autotaxin inhibitory activity, LPA receptor-activating action, and ERK-phosphorylating action.
  • the present invention has been completed based on the above-described findings.
  • a method for inhibiting autotaxin comprising administering the compound represented by the formula (1) to a target (subject).
  • a method for activating an LPA receptor comprising administering the compound represented by the formula (1) to a target (subject).
  • a method for phosphorylating ERK comprising administering the compound represented by the formula (1) to a target (subject).
  • novel carbalysophosphatidic acid is provided.
  • the carbalysophosphatidic acid of the present invention has autotaxin inhibitory activity, LPA receptor-activating action, and ERK-phosphorylating action.
  • the present invention relates to a compound represented by the following formula (1): wherein R represents a linear or branched alkyl group containing 1 to 30 carbon atoms, a linear or branched alkenyl group containing 2 to 30 carbon atoms, or a linear or branched alkynyl group containing 2 to 30 carbon atoms, and these groups may optionally comprise a cycloalkane ring or an aromatic ring; and M represents a hydrogen atom or a counter cation.
  • R represents a linear or branched alkyl group containing 1 to 30 carbon atoms, a linear or branched alkenyl group containing 2 to 30 carbon atoms, or a linear or branched alkynyl group containing 2 to 30 carbon atoms, and these groups may optionally comprise a cycloalkane ring or an aromatic ring
  • M represents a hydrogen atom or a counter cation.
  • linear or branched alkyl group containing 1 to 30 carbon atoms represented by the substituent R in the formula (1) may include a methyl group, an ethyl group, a propyl group, a butyl group, a pentyl group, a hexyl group, a heptyl group, an octyl group, a nonyl group, a decyl group, an undecyl group, a dodecyl group, a tridecyl group, a tetradecyl group, a pentadecyl group, a hexadecyl group, a heptadecyl group, an octadecyl group, a nonadecyl group, and an eicosyl group.
  • linear or branched alkenyl group containing 2 to 30 carbon atoms represented by the substituent R may include an allyl group, a butenyl group, an octenyl group, a decenyl group, a dodecadienyl group, and a hexadecatrienyl group.
  • More specific examples thereof may include an 8-decenyl group, an 8-undecenyl group, an 8-dodecenyl group, an 8-tridecenyl group, an 8-tetradecenyl group, an 8-pentadecenyl group, an 8-hexadecenyl group, an 8-heptadecenyl group, an 8-octadecenyl group, an 8-icosenyl group, an 8-docosenyl group, a heptadeca-8,11-dienyl group, a heptadeca-8,11,14-trienyl group, a nonadeca-4,7,10,13-tetraenyl group, a nonadeca-4,7,10,13,16-pentaenyl group, and a henicosa-3,6,9,12,15,18-hexaenyl group.
  • linear or branched alkynyl group containing 2 to 30 carbon atoms represented by the substituent R may include an 8-decynyl group, an 8-undecynyl group, an 8-dodecynyl group, an 8-tridecynyl group, an 8-tetradecynyl group, an 8-pentadecynyl group, an 8-hexadecynyl group, an 8-heptadecynyl group, an 8-octadecynyl group, an 8-icosynyl group, an 8-docosynyl group, and a heptadeca-8,11-diynyl group.
  • cycloalkane ring optionally contained in the above-described alkyl group, alkenyl group or alkynyl group may include a cyclopropane ring, a cyclobutane ring, a cyclopentane ring, a cyclohexane ring, and a cyclooctane ring.
  • the cycloalkane ring may optionally contain one or more heteroatoms, and examples of such a cycloalkane ring may include an oxirane ring, an oxetane ring, a tetrahydrofuran ring, and an N-methylprolidine ring.
  • aromatic ring optionally contained in the above-described alkyl group, alkenyl group or alkynyl group may include a benzene ring, a naphthalene ring, a pyridine ring, a furan ring, and a thiophene ring.
  • substituent R that is an alkyl group substituted with a cycloalkane ring may include a cyclopropylmethyl group, a cyclohexylethyl group, and an 8,9-methanopentadecyl group.
  • substituent R that is an alkyl group substituted with an aromatic ring may include a benzyl group, a phenethyl group, and a p-pentylphenyloctyl group.
  • R is preferably a linear or branched alkyl group containing 9 to 17 carbon atoms, a linear or branched alkenyl group containing 9 to 17 carbon atoms, or a linear or branched alkynyl group containing 9 to 17 carbon atoms.
  • R is more preferably a linear or branched alkyl group containing 9, 11, 13, 15 or 17 carbon atoms, or a linear or branched alkenyl group containing 9, 11, 13, 15 or 17 carbon atoms.
  • R is particularly preferably a linear or branched alkenyl group containing 9, 11, 13, 15 or 17 carbon atoms.
  • -CO-R is a palmitoyl group or an oleoyl group.
  • M in the formula (1) is a hydrogen atom or a counter cation.
  • M that is a counter cation may include an alkali metal atom, an alkaline-earth metal atom, and a substituted or unsubstituted ammonium group.
  • the alkali metal atom may include lithium, sodium, and potassium.
  • the alkaline-earth metal atom may include magnesium and calcium.
  • the substituted ammonium group may include a butylammonium group, a triethylammonium group, and a tetramethylammonium group.
  • the compound represented by the formula (1) includes isomers such as positional isomers, geometric isomers, tautomers, or optical isomers, depending on the type of the substituent thereof. All possible isomers and mixtures comprising two or more types of the isomers at any given ratio are also included in the scope of the present invention.
  • the compound represented by the formula (1) may be present in the form of an adduct with water or various types of solvents (i.e. a hydrate or a solvate) in some cases. These adducts are also included in the scope of the present invention. In addition, any given crystal forms of the compound represented by the formula (1) and a salt thereof are also included in the scope of the present invention.
  • the compound represented by the formula (1) can be produced, for example, according to the method described in the after-mentioned Example 1 or Example 2.
  • a solution is prepared by dissolving a carbacyclic phosphatidic acid derivative having the following structure in a suitable buffer (for example, a phosphate buffer, etc.): wherein R and M are the same as those defined in the formula (1).
  • a suitable buffer for example, a phosphate buffer, etc.
  • R and M are the same as those defined in the formula (1).
  • the thus prepared solution is diluted with an artificial gastric juice (for example, a sodium chloride aqueous solution (2 g of NaCl, 7 mL of 12 M HCl/1000 mL of water, pH 1.2)).
  • an artificial gastric juice for example, a sodium chloride aqueous solution (2 g of NaCl, 7 mL of 12 M HCl/1000 mL of water, pH 1.2
  • the obtained solution is incubated, so that the compound represented by the formula (1) can be produced.
  • the compound represented by the formula (1) can be produced using autotaxin (ATX).
  • ATX and a carbacyclic phosphatidic acid derivative having the above-described structure are dissolved in a suitable buffer (for example, a Tris buffer, etc.) to result in predetermined concentrations.
  • a suitable buffer for example, a Tris buffer, etc.
  • the obtained solution is reacted by incubation at 37°C, so that the compound represented by the formula (1) can be produced.
  • the compound represented by the formula (1) of the present invention can be used as an autotaxin inhibitor, an LPA receptor activator, or an ERK-phosphorylating agent.
  • ATX is a phospholipid metabolizing enzyme, which decomposes lysophosphatidylcholine (LPC) and generates lysophosphatidic acid (LPA).
  • Lysophosphatidic acid is one of lipid mediators associated with signal transduction, and binds to LPA receptors (6 types of G protein-coupled receptors).
  • ERK Extracellular Signal-regulated Kinase
  • MAPK Extracellular Signal-regulated Kinase
  • EGFR epidermal growth factor receptor
  • the autotaxin inhibitor, LPA receptor activator or ERK-phosphorylating agent according to the present invention may be provided in the form of a reagent composition or a pharmaceutical composition, comprising the compound represented by the formula (1) as an active ingredient and one or two or more pharmaceutically acceptable preparation additives.
  • the autotaxin inhibitor, LPA receptor activator or ERK-phosphorylating agent of the present invention can be administered in various forms.
  • a preferred administration form may be either oral administration or parenteral administration (e.g. intravenous, intramuscular, subcutaneous or intradermal injection, intrarectal administration, transmucosal administration, etc.).
  • parenteral administration e.g. intravenous, intramuscular, subcutaneous or intradermal injection, intrarectal administration, transmucosal administration, etc.
  • a pharmaceutical composition suitable for oral administration may include a tablet, a granule, a capsule, a powder agent, a solution agent, a suspension, and a syrup.
  • Examples of a pharmaceutical composition suitable for parenteral administration may include an injection, drops, a suppository, and a transdermal absorbent.
  • the dosage form of the autotaxin inhibitor, LPA receptor activator or ERK-phosphorylating agent of the present invention is not limited thereto.
  • a prolonged action preparation can be produced using the compound of the present invention according to a known technique.
  • the compound of the present invention serving as an active ingredient is enclosed into hydrogel comprising gelatin as a base material, so as to produce a sustained release preparation.
  • preparation additives which are used in production of the autotaxin inhibitor, LPA receptor activator or ERK-phosphorylating agent of the present invention, are not particularly limited, and can be appropriately selected by a person skilled in the art.
  • preparation additives that can be used herein may include excipients, disintegrators or disintegration aids, binders, lubricants, coating agents, base materials, solubilizers or solubilizing agents, dispersants, suspending agents, emulsions, buffer agents, antioxidants, antiseptics, tonicity agents, pH adjusters, solubilizers, and stabilizers. Individual specific components used for these purposes are publicly known to such a skilled person.
  • preparation additives that can be used in production of a preparation for use in oral administration may include: excipients, such as glucose, lactose, D-mannitol, starch, or crystalline cellulose; disintegrators or disintegration aids, such as carboxymethyl cellulose, starch, or calcium carboxymethyl cellulose; binders, such as hydroxypropyl cellulose, hydroxypropylmethyl cellulose, polyvinyl pyrrolidone, or gelatin; lubricants, such as magnesium stearate or talc; coating agents, such as hydroxypropylmethyl cellulose, white sugar, polyethylene glycol, or titanium oxide; and base materials, such as petrolatum, liquid paraffin, polyethylene glycol, gelatin, kaolin, glycerin, purified water, or hard fat.
  • excipients such as glucose, lactose, D-mannitol, starch, or crystalline cellulose
  • disintegrators or disintegration aids such as carboxymethyl cellulose, starch, or
  • preparation additives that can be used in production of a preparation for use in injections or drops may include: solubilizers or solubilizing agents that may constitute aqueous injections or use-time dissolution type injections, such as distilled water, a normal saline, propylene glycol, and a surfactant, which are for use in injections; tonicity agents, such as glucose, sodium chloride, D-mannitol, and glycerin; and pH adjusters, such as inorganic acids, organic acids, inorganic bases, or organic bases.
  • the autotaxin inhibitor, LPA receptor activator or ERK-phosphorylating agent of the present invention can be administered to mammals such as humans.
  • the applied dose of the autotaxin inhibitor, LPA receptor activator or ERK-phosphorylating agent of the present invention should be increased or decreased, as appropriate, depending on conditions such as the age, sex and body weight of a patient, symptoms, and an administration route.
  • the daily dose of an active ingredient per adult is in the range of approximately 1 ⁇ g/kg to approximately 1,000 mg/kg, and is preferably in the range of approximately 10 ⁇ g/kg to approximately 100 mg/kg.
  • the above-described dose of drug may be administered once or divided over several administrations (e.g. about 2 to 4 times) per day.
  • 2ccPA (5 mg) was dissolved in 1 mL of a phosphate buffer, and a 2ccPA/PBS solution was then diluted with the artificial gastric juice described in the Japanese Pharmacopoeia, 17th Editi on (a sodium chloride aqueous solution (2 g of NaCl, 7 mL of 12 M HCl/1000 mL of water, pH 1.2) to result in a final concentration of 1 mg/mL. Thereafter, this solution was incubated at 37°C for 10, 30, or 60 minutes.
  • a copper acetate-phosphoric acid reagent 3% copper(II) acetate monohydrate (w/v), 8% phosphoric acid (v/v), and 2% sulfuric acid (v/v) was sprayed onto the plate, and the plate was then heated at 150°C for 5 minutes, so that the spots of the compound could be visualized ( Fig. 1 ).
  • 2ccPA (10 mg) was dissolved in 2 mL of the above-described artificial gastric juice (a sodium chloride aqueous solution (2 g of NaCl, 7 mL of 12 M HCl/1000 mL of water, pH 1.2). Thereafter, the temperature of this solution was set at 37°C, and 2 hours later, 8 mL of chloroform : methanol (2 : 1, v/v) was added to the solution. The solution was suspended, and was then separated into two layers according to centrifugation (1500 g x 5 minutes). Thereafter, a lower layer was fractionated.
  • a sodium chloride aqueous solution (2 g of NaCl, 7 mL of 12 M HCl/1000 mL of water, pH 1.2.
  • the recovered supernatant was dried with nitrogen, and was then dissolved in 1 mL of methanol, followed by filtration through a filter (0.2 ⁇ m Captiva Premium syringe filter, Agilent Technologies). The filtrate was dried with nitrogen, so as to obtain approximately 4.2 mg of a compound with an Rf value of about 0.29.
  • the solution was developed onto a thin-layer chromatography plate, and a portion of the plate was then cut off. Thereafter, a copper acetate-phosphoric acid reagent (3% copper(II) acetate monohydrate (w/v), 8% phosphoric acid (v/v), and 2% sulfuric acid (v/v)) was sprayed onto the cut plate portion, and the plate portion was then heated at 150°C for 5 minutes, so that the spot could be visualized and identified.
  • a copper acetate-phosphoric acid reagent 3% copper(II) acetate monohydrate (w/v), 8% phosphoric acid (v/v), and 2% sulfuric acid (v/v)
  • the compound (approximately 4.2 mg) obtained in the above (2) was suspended in 10 mL of methanol, and the obtained solution was 100-fold diluted with 5 mM ammonium formate-containing methanol/water (95 : 5, v/v), so as to obtain a sample for Sciex Triple TOF4600 (QqTOF) mass spectrometer (measurement conditions will be shown in the following table).
  • QqTOF Sciex Triple TOF4600
  • Table 1 QqTOF conditions Items Conditions Ion source temperature 0°C Curtain gas 20 Source gas 1 15 Source gas 2 0 Ion spray voltage -4500 V Declustering potential -100 V Scanning range (Cycle time, collision energy) Full ion scan ⁇ m/z: 50-500 (250 ms, -10 eV) Product ion scan ⁇ m/z: 50-500 (100 ms, -30 or -75 eV)
  • ATX (Cayman chemical, MI) and 2ccPA were dissolved in 40 ⁇ L of a Tris buffer (50 mM Tris-HCl, pH 8.0, 140 mM NaCl, 5 mM KCl, 1 mM CaCl 2 , and 1 mM MgCh), to result in final concentrations of 50 nM and 10 ⁇ M, respectively. Thereafter, the mixed solution was reacted at 37°C for 0, 2, and 4 hours. After completion of the reaction, 160 ⁇ L of acetic methanol (pH 4.0) was added to and fully suspended in the reaction solution.
  • a Tris buffer 50 mM Tris-HCl, pH 8.0, 140 mM NaCl, 5 mM KCl, 1 mM CaCl 2 , and 1 mM MgCh
  • ATX and LPC 16:0 were suspended in 40 ⁇ L of a Tris buffer (50 mM Tris-HCl, pH 8.0, 140 mM NaCl, 5 mM KCl, 1 mM CaCl 2 , and 1 mM MgCl 2 ) to result in final concentrations of 50 nM and 10 ⁇ M, respectively.
  • 2ccPA or 2carbaLPA was suspended in the aforementioned Tris buffer to result in a final concentration of 10 ⁇ M. Thereafter, the obtained mixture was incubated at 37°C. At each reaction time, 160 ⁇ L of acidic methanol was added to and fully suspended in the reaction solution.
  • the amount of LPA 16:0 that came to be synthesized at each reaction time was measured.
  • LPA was synthesized in a time-dependent manner in the reaction solution containing neither 2ccPA nor 2carbaLPA, whereas the synthesis of LPA was suppressed in the reaction solution containing 2ccPA or 2carbaLPA ( Fig. 6 ).
  • ATX and LPC 16:0 were dissolved in 100 ⁇ L of a choline oxidase reaction solution (0.05% 4-aminoantipyrine, 0.05% TOOS (N-Ethyl-N-(2-hydroxy-3-sulfopropyl)-3-methylaniline, sodium salt, and dihydrate), 1 unit of peroxidase, 1 unit of choline oxidase, 50 mM Tris-HCl, pH 8.0, 140 mM NaCl, 5 mM KCl, 1 mM CaCl 2 , and 1 mM MgCl 2 )to result in final concentrations of 50 nM and300 ⁇ M, respectively.
  • a choline oxidase reaction solution 0.05% 4-aminoantipyrine, 0.05% TOOS (N-Ethyl-N-(2-hydroxy-3-sulfopropyl)-3-methylaniline, sodium salt, and dihydrate
  • 1 unit of peroxidase 1 unit of choline
  • SW1353 cells were seeded at a density of 3 x 10 4 cells/well on a 6-well plate.
  • the cell culture solution was exchanged with a serum-free culture solution, and the cells were cultured for 6 hours. Thereafter, 2carbaLPA dissolved in 0.1% BSA/PBS or 0.1% BSA/PBS (Vehicle) was added to the culture to a final concentration of 10 ⁇ M.
  • the obtained mixture was incubated at 37°C for 30 minutes, and the cells were then recovered using a sample buffer (0.125 M Tris-HCl, pH 6.8, 4% SDS, 20% glycerol, 0.01% BPB, and10% 2-mercaptoethanol.
  • the protein was separated by SDS-PAGE, and the protein in the SDS-PAGE gel was transcribed onto a PVDF membrane according to a Western-blotting method.
  • the transcribed protein was detected using an anti-pERK antibody, an anti-ERK antibody and an anti-P-tubulin antibody as primary antibodies, and using an anti-rabbit IgG antibody as a secondary antibody.
  • the band of pERK became dense, and thus, it was demonstrated that 2carbaLPA can phosphorylate ERK.
  • 2ccPA (19.70 ⁇ 1.39 mg) was enclosed in a capsule (size 9, Torpac Inc., Fairfield, NJ)).
  • the capsule was coated with Eudragit S100/ L100 (4/1, 11 mg/cm 2 , pH 6.8) (Evonik Japan Co., Ltd., Tsukuba, Japan).
  • the thus obtained capsule was orally administered to 7-week-old SD rats (body weight: 190 to 220 g), into each of which a cannula had been transvenously inserted in advance, using a capsule injector (Natsume Seisakusho, Tokyo, Japan).
  • the cannula-inserted rats were purchased from Oriental Yeast Co., Ltd.
  • Plasma sample was extracted from the plasma sample in the same manner as that described in the study paper ( Journal of Chromatography B 1076 (2016) 15-21 , Quantitative determination of cyclic phosphatidic acid and its carba analog in mouse organs and plasma using LC-MS/MS).
  • HEK293A or ALPA 2/5/6 HEK293A was suspended in a Dulbecco-modified Eagle's medium (DMEM supplemented with 10% fetal calf serum, 100 U/mL penicillin, and 100 ⁇ g/mL streptomycin) to result in a concentration of 2 x 10 5 cells/mL, and the thus obtained suspension was then seeded on a dish having a diameter of 100 mm (10 mL/dish). Thereafter, the cells were incubated at 37°C in 5% CO 2 for 24 hours.
  • DMEM Dulbecco-modified Eagle's medium
  • Plasmid solution Opti-MEM (Thermo Fischer Scientific) 500 ⁇ L Alkaline phosphatase labeled TGF ⁇ . (AP-TGF ⁇ ) expression vector 2.5 ⁇ g GPCR expression vector 1 ⁇ g
  • Mock-transfected cells were transfected with an empty vector, instead of a GPCR expression vector.
  • LPA 1 - and LPAs-expressing cells and control cells thereof were also transfected with a G ⁇ q/i1 expression vector (0.5 ⁇ g/dish).
  • LPA 2 - and LPA 4 -expressing cells and control cells thereof were also transfected with a G ⁇ q/s expression vector (0.5 ⁇ g/dish).
  • HEK293A cells were used for the expression of LPA 1 and LPA 4 .
  • ⁇ LPA 2/5/6 HEK293A cells were used for the expression of LPA 2 , LPA 3 , LPA 5 , and LPA 6 .
  • human genes were used for all LPA receptors.
  • HEK293A and ⁇ LPA 2/5/6 HEK293A cells were washed with PBS.
  • the resulting cells were peeled with 0.05% Trypsin/0.53 mM EDTA (2 mL/dish), and were then neutralized with DMEM (3 mL/dish).
  • the cells were centrifuged (190 x g, 5 min), and were then re-suspended in a Hank's balanced salt solution (HBSS supplemented with 5 mM HEPES (pH 7.4)) (10 mL/dish), followed by incubation at room temperature for 15 minutes.
  • HBSS Hank's balanced salt solution
  • HEPES pH 7.4
  • the cells were re-suspended in HBSS, and were then seeded on a 96-well plate (cell plate) (LPA 1 -, LPA 2 -, and LPA 3 -expressing cells, and the mock-transfected cells thereof: 90 ⁇ L/well; LPA 4 -, LPA 5 -, and LPA 6 -expressing cells and the mock-transfected cells thereof: 80 ⁇ L/well, 1-2 x 10 4 cells/well).
  • the cells were incubated at 37°C in 5% CO 2 for 30 minutes, and an agonist (2ccPA or 2carbaLPA) was added thereto in a concentration 10 times higher than the final concentration (10 ⁇ L/well; the compound was diluted with HBSS supplemented with 0.01% bovine serum albumin).
  • an agonist (2ccPA or 2carbaLPA) was added thereto in a concentration 10 times higher than the final concentration (10 ⁇ L/well; the compound was diluted with HBSS supplemented with 0.01% bovine serum albumin).
  • 100 ⁇ M Ki 16425 was added 5 minutes before addition of the agonist (10 ⁇ L/well).
  • the cells were incubated at 37°C in 5% CO 2 for 60 minutes, and the plate was then centrifuged (190 x g, 2 min). Thereafter, the obtained supernatant was transferred into another 96-well plate (supernatant plate) (80 ⁇ L/well).
  • 1M p -nitrophenyl phosphate (containing 120 mM Tris-HCl (pH 9.5), 40 mM NaCl, and 10 mM MgCl 2 ) was dispensed in individual wells of the supernatant plate and the cell plate (80 ⁇ L/well). Immediately after the dispensing and 1 hour after the dispensing, OD405 was measured.
  • EC 50 and E max were calculated by fitting the data to a 4-parameter logistic curve, using Graphpad Prism 6 (Graphpad).
  • 2carbaLPA exhibited agonist activity against all of the LPA receptors (LPA 1 , LPA 2 , LPA 3 , LPA 4 , LPA 5 , and LPA 6 ).
  • the agonist activities (EC 50 ) of 2carbaLPA against LPA 1 , LPA 2 , LPA 3 , LPA 4 , LPA 5 , and LPA 6 were 10 nM, 90 nM, 1.2 nM, 0.7 nM, 27 nM, and 43 nM, respectively.

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EP20890320.3A 2019-11-22 2020-11-20 Acide carbalysophosphatidique Pending EP4062976A4 (fr)

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JP2019211367A JP6727596B1 (ja) 2019-11-22 2019-11-22 カルバリゾホスファチジン酸
PCT/JP2020/043362 WO2021100847A1 (fr) 2019-11-22 2020-11-20 Acide carbalysophosphatidique

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JP4815063B2 (ja) 2001-04-13 2011-11-16 きみ子 室伏 環状ホスファチジン酸を含むグリア細胞の増殖、分化及び／又は生存の促進のための薬剤
US20040214799A1 (en) 2001-05-21 2004-10-28 Mutsuko Mukai Cancerous metastasis inhibitors containing carbacyclic phosphatidic acid derivatives
WO2008081580A1 (fr) 2006-12-28 2008-07-10 Ochanomizu University Agent analgésique comprenant un dérivé cyclique d'acide phosphatidique
JP2011211921A (ja) * 2010-03-31 2011-10-27 Sansho Kk 環状ホスファチジン酸の製造方法
JP5737888B2 (ja) 2010-09-06 2015-06-17 Ｓａｎｓｈｏ株式会社 アトピー性皮膚炎治療剤
US10413559B2 (en) 2013-01-28 2019-09-17 Ochanomizu University Method for treating demyelinating disease
JP6864899B2 (ja) * 2016-11-14 2021-04-28 国立大学法人お茶の水女子大学 損傷治療剤

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US20230108750A1 (en) 2023-04-06

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